The Study Of Molecular Mechanisms Of Cyclosporine A Inhibiting Insulin Secretion

Objective Post-transplant diabetes mellitus(PTDM)is a common complication of organ transplantation.One of the most important risk factors is the immunosuppressive regimen.The diabetogenicity of calcineurin inhibitors,including cyclosporine A,have been demonstrated to be mediated by insulin resistance,insulin secretion impairment and apoptosis of pancreatic ? cells.Our previous studies have indicated that a mini p-glycoprotein(encoded by the p-glycoprotein gene abcb1),regulated the release of insulin granules.CsA,as the specific inhibitor of p-glycoprotein,could inhibit insulin secretion through its biological functions to p-glycoprotein.In this study,we explored the underlying mechanisms of CsA inhibiting insulin release in vitro and in vivo.In addition,the potentials of mini p-glycoprotein involving in the pathogenesis of PTDM were preliminary investigated.Methods in vitro:(1)Rat islets were isolated from pancreas by collagenase ?digestion.The expressions of mini p-glycoprotein of a and ? cells were observed using a Zeiss LSM 510 confocal microscope.(2)The islets were stained by AO/PI and evaluated with fluorescence microscope after inoculations of 0.5?g/ml?1.0?g/ml?2.5?g/ml?5.0?g/ml?1O?g/ml CsA for 6 hours,24 hours and 48 hours,respectively.The islets treated only by the vehicle were served as control.(3)Islets were treated by different concentrations of CsA(1?g/ml and 5?g/ml)or the vehicle for 6 hours and 24 hours,respectively.The biphasic insulin release was measured by radioimmunology(RIA)assay after static incubations of the islets in low and high glucose buffers.(4)The expressions of abcb1b,pdx1,ins1,ins2,glucagon,casp3 and Bcl-2 were determined by real-time fluorescence quantitative PCR.in vivo:(1)Rat were injected once a day with saline(control group)or CsA(10mg/kg,CsA group)subcutaneously.After 2 weeks,the animals were subjected to an intraperitoneal glucose tolerance test(2.0g/kg BW)and the blood glucose were measured(tail vein)just before and after glucose challenge at several time points.(2)The pathological examination of the pancreases were carried out by routine lab methods.(3)The changes of basal and high glucose stimulated insulin secretion were evaluated though in situ pancreas perfusions.(4)The expressions of abcb1b,ins1,ins2,glucagon,casp3 and Bcl-2 were determined by real-time fluorescence quantitative PCR.Results in vitro:(1)Mini p-glycoprotein mainly expressed in the B cells.The overlay studies indicated that mini p-glycoprotein was colocalized with insulin partially.(2)The islets incubated with 0.5?g/ml and 1.0?tg/ml CsA did not show any differences compared to the control group.Treated with 2.5?g/ml CsA for 48 hours,the red fluorescent increased significantly among the islet cells,but the islets kept the round and smooth shapes.The islets became irregular and broken after exposed to 5.0?g/ml and 10?gml CsA for 24 hours,respectively.The islets were broken totally and formed debris after 48 hours incubation of 10?g/ml CsA.(3)After incubation of CsA(1.0?g/ml)for 6 or 24 hours,the second phases of insulin secretion in both treatment were reduced,but the first phases were not influenced.(4)The expressions of abcb1b,insl,ins2,pdx1 and glucagon did not show any differences after 6 and 24 hours incubation of CsA(1.0?g/ml),respectively.The apoptosis genes were not changed after incubation of CsA(1.0?g/ml)for 6 hours,but the expression of Bcl-2 was down-regulated after 24 hours incubation.in vivo:(1)The levels of blood glucose at 30 min were higher in the treated group than that of the control group,whereas the levels of blood glucose at other time points(basal,60min and 120min)did not show any differences compare to those of the control group.The pathological slides showed that no significant injuries were observed in the rat pancreases treated by CsA.Both the first and second phase of insulin secretion stimulated by high glucose were reduced significantly in the perfused pancreases;but the basal insulin secretion in both groups(control vs CsA)were in similar levels.The Bcl-2 gene was down-regulated in CsA group.The other detected genes(abcb1b,ins1,ins2,glucagon and casp3)did not show any difference between the two groups.Conclusion(1)Mini p-glycoprotein mainly expresses in islet B cells,probably localize in insulin granules.(2)CsA has direct toxic effects to islets in dose and time-dependent manner.The suppression of insulin secretion of CsA is not induced by reduction of insulin biogenesis and apoptosis when the islets exposed to low dosage of CsA in short period.CsA might affect biphasic insulin secretion throuth the mini p-glycoprotein pathway.This effect might be mainly due to pharmacological inhibition rather than the influence of mini p-glycoprotein expression(3)CsA does not cause significantly injury to the pancreases of rats treated with CsA in vivo.CsA inhibits biphasic insulin secretion significantly.One of the principal pathogenesis may be apoptosis pathway.(4)CsA suppresses insulin release through several pathways,of which cause PTDM.(5)In fact that CsA can inhibit biphasic insulin secretion,we should be cautious in its clinical applications to diabetic patients who possess partially insulin secretion ability,in particular the second phase secretion.